CN108130349A - A kind of synthetic method of the oligosaccharides of the mono- aggressiveness of Lewis x, dimer and its sialylated derivative - Google Patents
A kind of synthetic method of the oligosaccharides of the mono- aggressiveness of Lewis x, dimer and its sialylated derivative Download PDFInfo
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Abstract
The invention discloses the synthetic methods of the oligosaccharides of a kind of mono- aggressiveness of Lewis x, dimer and its sialylated derivative.Including：Core skeleton compound (V)~(IX) is built using enzyme process modularization assembling 1 and enzyme process modularization assembling 2；Enzyme process modularization assembling 3 is recycled to build LexSingle aggressiveness, dimer (I)~(II)；Finally sLe is built using enzyme process modularization assembling 4xSingle aggressiveness, dimer (III)~(IV).The present invention combines regioselectivity high possessed by enzymatic clarification and high efficiency, and the synthesis of target molecule is realized with higher yield.And glycosyl transferase, riboside generation enzyme and the sugared kinases utilized in the present invention is prokaryotes source, has many advantages, such as that expressing quantity is high, substrate adaptability is wide, high catalytic efficiency, available for largely preparing.
Description
Technical field
Synthetic method more particularly to a kind of Lewis x (Le the present invention relates to glucidex) single aggressiveness, dimer and
The synthetic method of the oligosaccharides of its sialylated derivative.
Background technology
Lewis x[Lex, Gal β l-4 (Fuc α 1-3) GlcNAc β OR] and antigen (as shown in Figure 1) and sialylated Lewis
x[sLex, Neu5Ac α 2-3Gal β l-4 (Fuc α 1-3) GlcNAc β OR] and antigen (as shown in Figure 2) is nuclear structure type II
(Gal β l-4Glc-NAc β OR) (as shown in Figure 3) modifies obtained oligosaccharides through α 1-3 are fucosylated and α 2-3 are sialylated, extensively
It is present in many N-/O- glycan of human milk oligosaccharides, bacteria lipopolysaccharide, glycolipid and mammalian cell expression.They are in biology
Key effect is played in the biometric identification process that weight is wanted, such as promotes the growth of beneficial bacterium in intestinal flora, promotes cell
Middle signal transduction, cell adherence, metastases prevent pathogenic infection, adjust intestinal epithelial cell response, immunological regulation etc..
Numerous studies show Lex、sLexAntigen can be combined as host cell surface receptor with microorganism or toxin, so as to prevent
The combination of pathogen and intestinal epithelial cell prevents the generation of disease.Such as cause the helicobacter pylori of chronic gastritis
Helicobacter pylori can combine sLexOligosaccharides, so as to which pathogen be inhibited to stick host cell.In addition, contain
LexStructure and sLexThe high expression in many tumour cells of the oligosaccharides of structure, is referred to as Tumor associated carbohydrate antigen (TACAs), it
Be cell adhesion molecule (cell adhesion molecular, CAM) family important member, in tumour cell and host
Key effect is played in Heterogeneity adhesion between cell, between the cell and cell of mediation, between cell and extracellular matrix
Interaction be the key that malignant tumour invasion transfer.Therefore it is closely related with the generation of tumour, development, often by as tumour
The marker of diagnosis and the prognostic indicator of metastases, content in blood can as differentiate innocent and malignant tumour it is important according to
According to.
Substantial connection just because of TACAs and tumour and its characteristics of be mainly distributed on tumour cell, provides swollen
The new approaches of knurl treatment, stimulate the immune system of body using TACAs antigen, immune system are made to identify and generate and is directed to
The antibody of TACAs has the anti-tumor vaccine of targeting with this, the treatment applied to cancer.
In addition, LexAnd sLexAntigen can be combined with carbohydrate-binding protein (glycan-binding proteins, GBPs), and two
The interaction of person shows multiple functions in tumour cell immunoregulation network, such as inflammation, immunosupress, cell is promoted to wither
It dies.Therefore it designs, synthesize Substrate competitive inhibitors or prepare compatibility monoclonal antibody to block the combination of the two, be tumour
Diagnose and treat provide a new solution route.
In view of the foregoing, Le is largely synthesizedxAnd sLexAntigen excavates its potential sugar chain composition library, develop using its as
Anti-inflammatory, the antitumor newtype drug and novel early diagnosis reagent of lead compound are of great significance.
Since biological vivo environment is sufficiently complex, for current research level, in a manner of extraction out of organism
It is very little and abnormal difficult to obtain possibility of the single human milk oligosaccharides of a large amount of structures for biology, study of pharmacy.For
For chemical synthesis, due to the similar polyhydroxy structure of the activity of sugar chain inherently, guarantor repeatedly is needed in the synthesis process
Shield and deprotection operation ensure region, stereoselectivity, lead to that its reaction step is more, overall yield is low.
Invention content
In order to solve the deficiencies in the prior art, the object of the present invention is to provide a kind of fast and efficiently synthetic methods to solve
Certainly problem above.Purpose sugar chain is synthesized using enzyme process modularization assembling strategy, is system research LexAnd sLexAntigen as it is anti-inflammatory,
Application in terms of antitumor drug lays the foundation.
To achieve these goals, the present invention uses following technical proposals：
The first aspect of the invention provides a kind of synthetic method of core skeleton sugar chain 1,
1) N-acetylglucosamine β 1-3 glycosidic bonds are coupled to two shown in formula (V) using enzyme process modularization assembling 1
On sugar, the trisaccharide shown in formula (VI) is synthesized；
2) galactolipin is coupled to β 1-4 glycosidic bonds on the trisaccharide shown in formula (VI) using enzyme process modularization assembling 2, closed
Into the tetrose shown in formula (VII)；
Wherein, the R in formula (V)~(VII)1For hydroxyl, azide substitution alkyl, alkynyl substituted alkyl, sulfydryl replace alkyl,
α-or beta configuration substitution alkyl, α-or beta configuration serine residue, α-or beta configuration threonine residues；
The enzyme that modified N-acetylglucosamine obtains intermediate 1 in the enzyme process modularization assembling 1 is N- acetylaminos
The enzyme of glucokinase and riboside generation enzyme, catalytic intermediary 1 and polysaccharide synthesis is shifted for β 1-3N- acetylglucosamines
Enzyme；
The enzyme that modified galactolipin obtains intermediate 2 in the enzyme process modularization assembling 1 is galactokinase and riboside life
Into enzyme, the enzyme of catalytic intermediary 2 and polysaccharide synthesis is β 1-4 galactosyl transferases.
The second aspect of the invention provides a kind of synthetic method of core skeleton sugar chain 2,
(1) N-acetylglucosamine is coupled to β 1-3 glycosidic bonds shown in formula (VII) using enzyme process modularization assembling 1
Tetrose on, synthesize the pentasaccharides shown in formula (VIII)；
(2) galactolipin is coupled to β 1-4 glycosidic bonds on the pentasaccharides shown in formula (VIII) using enzyme process modularization assembling 2,
Six sugar shown in synthesis formula (IX)；
Wherein, the R in formula (VII)~(IX)1Replace alkane for hydroxyl, azide substitution alkyl, alkynyl substituted alkyl, sulfydryl
Base, α-or beta configuration substitution alkyl, α-or beta configuration serine residue, α-or beta configuration threonine residues；
The enzyme that modified N-acetylglucosamine obtains intermediate 3 in the enzyme process modularization assembling 1 is N- acetylaminos
The enzyme of glucokinase and riboside generation enzyme, catalytic intermediary 3 and polysaccharide synthesis is shifted for β 1-3N- acetylglucosamines
Enzyme；
The enzyme that modified galactolipin obtains intermediate 4 in the enzyme process modularization assembling 1 is galactokinase and riboside life
Into enzyme, the enzyme of catalytic intermediary 4 and polysaccharide synthesis is β 1-4 galactosyl transferases.
The third method of the present invention provides a kind of above method and is preparing the mono- aggressiveness of Lewis x, dimer or its saliva
Application in liquid acidification derivative.
The fourth aspect of the invention provides a kind of containing LexThe synthesis side of the sugar chain of single dimeric structure or dimeric structure
Fucose is coupled to tetrose shown in formula (VII) with α 1-3 glycosidic bonds using enzyme process modularization assembling 3, synthesizes formula (I) institute by method
The pentasaccharides shown；
Or, be coupled to fucose on six sugar shown in formula (IX) with α 1-3 glycosidic bonds using enzyme process modularization assembling 3, it closes
Into eight sugar shown in formula (II)；
Wherein, the R in formula (VII), formula (IX), formula (I)~(II)1For hydroxyl, azide substitution alkyl, alkynyl substituted alkane
Base, sulfydryl substitution alkyl, α-or beta configuration substitution alkyl, α-or beta configuration serine residue, α-or beta configuration threonine residues；
The enzyme that modified fucose obtains intermediate 5 in the enzyme process modularization assembling 3 is Fucokinase and riboside life
Into enzyme, the enzyme of catalytic intermediary 5 and polysaccharide synthesis is α 1-3 fucosyl transferases.
The fifth aspect of the invention provides a kind of containing sLexThe synthesis of the sugar chain of single dimeric structure or dimeric structure
Sialic acid is coupled to pentasaccharides shown in formula (I), parallel projects formula by method using enzyme process modularization assembling 4 with α 2-3 glycosidic bonds
(III) six sugar shown in；
Or, be coupled to sialic acid on eight sugar shown in formula (II) with α 2-3 glycosidic bonds using enzyme process modularization assembling 4, it closes
Into nine sugar shown in formula (IV)；
Wherein, the R in formula (I)~(IV)1For hydroxyl, azide substitution alkyl, alkynyl substituted alkyl, sulfydryl replace alkyl,
α-or beta configuration substitution alkyl, α-or beta configuration serine residue, α-or beta configuration threonine residues；
The enzyme that modified sialic acid obtains intermediate 6 in the enzyme process modularization assembling 4 is riboside generation enzyme, and catalysis is intermediate
Body 6 and the enzyme of polysaccharide synthesis are α 2-3 fucosyl transferases.
It should be noted that the biosynthetic process of sugar chain drives process for non-template, be in a variety of glycosyl transferases and
Catalysis assembles under the collective effect of glycosidase, this causes the sugar chain structure of biofilm surface to show height inhomogeneity,
Therefore it is difficult to obtain enough, uniform and structure determination sugar chain and glycoconjugate from nature, this also becomes sugar synthesis and sugared work(
The bottlenecks of energy Journal of Sex Research.Although enzyme process modularization assembling strategy is a kind of synthetic method of efficient complex oligosaccharide,
It for different synthesis targets, needs targetedly to formulate synthesis strategy, is not simply under the catalysis of different enzymes, incites somebody to action
Different monosaccharide are simply connected together.
The present invention is for synthesis Lewis x (Lex) and sialylated Lewis x (sLex) single aggressiveness, dimer oligosaccharides set
Enzyme process modularization assembling strategy is counted.First, the target molecule being fitted to is analyzed, the composition of monosaccharide, the order of connection
And glycosidic bond configuration, it is determined that the synthesis required key enzyme of target oligosaccharides；Then screening, root key enzyme is optimized
According to the catalytic activity of enzyme, enzyme to factors such as regioselectivity, the stereoselectivities of substrate, different oligosaccharides point are targetedly built
The required enzyme process module of son；Finally probed into enzyme process react most suitable metal ion, concentration of substrate, reaction temperature, the reaction time,
System pH etc., ensure that the high-effect performance of enzyme in enzyme process module of the present invention, and finally realize the quick, efficient of target molecule
Synthesis.
Beneficial effects of the present invention：
1. the present invention dexterously combines the riboside generation enzyme and sugar of bacterial origin by enzyme process modularization assembling strategy
Based transferase realizes the Le with important biomolecule activityxAnd sLexSingle aggressiveness, dimer oligosaccharides preparation scale synthesis.This
The utilized glycosyl transferase of invention, riboside generation enzyme and sugared kinases are prokaryotes source, have expressing quantity
The advantages that height, substrate adaptability are extensive, high catalytic efficiency, thus the enzyme process modularization assembling based on it is efficient, and suitable
It is prepared in a large amount of；Expensive nucleosides can be efficiently converted into from monosaccharide that is inexpensive, being easy to get using ribotide generation enzyme
The glycosyl donor of activation, greatly reduces production cost；Relative to the relatively low chemical synthesis of complex steps, yield, enzyme process closes
Into having apparent advantage in terms of space and spatial chemistry specificity, reaction step is enormously simplified, improves the totality of reaction
Yield.
2. the efficient enzyme process modularization assembling strategy that the present invention develops is associated sugars structure and biological function research institute
The acquisition of sugar chain and its conjugate sample needed provides the high approach of feasibility, can also further investigate this on a molecular scale
The interaction mechanism and structure-activity relationship of a little sugar chains and receptor are controlled for illustrating for associated disease pathology mechanism with following diagnosis
Treatment is laid a good foundation.
Description of the drawings
The accompanying drawings which form a part of this application are used for providing further understanding of the present application, and the application's shows
Meaning property embodiment and its explanation do not form the improper restriction to the application for explaining the application.
Fig. 1：LexAntigen oligosaccharide structure；
Fig. 2：sLexAntigen oligosaccharide structure；
Fig. 3：Type II nuclear structures；
Fig. 4：The building-up process schematic diagram of enzyme process modularization assembling 1；
Fig. 5：The building-up process schematic diagram of enzyme process modularization assembling 2；
Fig. 6：The building-up process schematic diagram of enzyme process modularization assembling 3；
Fig. 7：The building-up process schematic diagram of enzyme process modularization assembling 4；
Fig. 8：The reaction equation of the lactose compound 1 of chemical method synthesis beta configuration；
Fig. 9：Enzyme process module is combined to the reaction equation of trisaccharide compound 2；
Figure 10：Enzyme process module is combined to the reaction equation of tetrose compound 3；
Figure 11：Enzyme process module is combined to the reaction equation of pentasaccharides compound 4；
Figure 12：Enzyme process module is combined to the reaction equation of six sugar compounds 5；
Figure 13：Enzyme process module is combined to the reaction equation of pentasaccharides compound 6；
Figure 14：Enzyme process module is combined to the reaction equation of eight sugar compounds 7.
Figure 15：Enzyme process module is combined to the reaction equation of six sugar compounds 8.
Figure 16：Enzyme process module is combined to the reaction equation of nine sugar compounds 9.
Specific embodiment
It is noted that described further below be all exemplary, it is intended to provide further instruction to the application.It is unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or combination thereof.
Enzyme process modularization assembling described herein is primarily referred to as being modified shape to monosaccharide using under the action of one group of enzyme
Into intermediate, intermediate is then made to carry out synthetic reaction with polysaccharide under the catalytic action of another group of enzyme, so that monosaccharide is even
It is coupled to polysaccharide.
Unless otherwise noted, the ratio of the amount of substance when " equivalent " in the application refers to matter interaction, such as：N-
The meaning of acetylglucosamine (1.2-5.0 equivalents) is：N-acetylglucosamine and the lactoside shown in formula (II)
The ratio of the amount of substance is 1.2-5.0.
As background technology is introduced, exist in the prior art that chemical synthesis process reaction step is more, overall yield is low
Deficiency, the technical issues of in order to solve as above, present applicant proposes a kind of mono- aggressiveness of Lewis x, dimer and its sialic acids
Change the synthetic method of the oligosaccharides of derivative.
A kind of exemplary embodiment of the application provides a kind of synthetic method of core skeleton sugar chain 1,
1) N-acetylglucosamine β 1-3 glycosidic bonds are coupled to two shown in formula (V) using enzyme process modularization assembling 1
On sugar, the trisaccharide shown in formula (VI) is synthesized；
2) galactolipin is coupled to β 1-4 glycosidic bonds on the trisaccharide shown in formula (VI) using enzyme process modularization assembling 2, closed
Into the tetrose shown in formula (VII)；
Wherein, the R in formula (V)~(VII)1For hydroxyl, azide substitution alkyl, alkynyl substituted alkyl, sulfydryl replace alkyl,
α-or beta configuration substitution alkyl, α-or beta configuration serine residue, α-or beta configuration threonine residues；
The enzyme that modified N-acetylglucosamine obtains intermediate 1 in the enzyme process modularization assembling 1 is N- acetylaminos
The enzyme of glucokinase and riboside generation enzyme, catalytic intermediary 1 and polysaccharide synthesis is shifted for β 1-3N- acetylglucosamines
Enzyme；
The enzyme that modified galactolipin obtains intermediate 2 in the enzyme process modularization assembling 1 is galactokinase and riboside life
Into enzyme, the enzyme of catalytic intermediary 2 and polysaccharide synthesis is β 1-4 galactosyl transferases.
Preferably, in the enzyme process modularization assembling 1, N-acetylglucosamine kinases is NahK, and riboside generates enzyme
For GlmU, β 1-3N- acetylglucosaminyl transferases are HpLgtA；
In the enzyme process modularization assembling 2, galactokinase GalK, riboside generates enzyme as BLUSP, β 1-4 galactolipins
Transferase is NmLgtB.
In the specific embodiment of the application, lactoside Gal β (l-4) GlcOR of the beta configuration shown in formula (V)1
Synthesis use following methods：After lactose is reacted with aceticanhydride, by it, all exposed hydroxyl is protected with acetyl group；Then it is sharp
Full acetylated lactose is subjected to beta configuration glycosylation reaction with microwave method, finally Azide and deprotection successively, you can obtain.
It can make the lactoside of lower step enzyme reaction receptor, specific reaction equation is as shown in Figure 8.
In the specific embodiment of the application, the synthetic method of the trisaccharide shown in formula (VI) is：It will be formula (V) Suo Shi
Lactoside, N-acetylglucosamine (1.2~5.0 equivalent), adenosine triphyosphate (ATP) (1.2~5.0 equivalent),
Uridine diphosphate guanosine triphosphate (UTP) (1.2~5.0 equivalent), MgCl2(5~100mmol), Tris-HCl buffer solutions (10-
500mmol, pH 5.0-10.0) obtained aqueous solution, then the pH value of reaction system is adjusted to 4.5~8.5, then adds N-
Acetylglucosamine kinases (NahK), UDP-N- acetylglucosamines generation enzyme (GlmU) and β 1-3N- acetylaminos Portugal
Grape sugar transferase (HpLgtA), reaction time are 3~72 hours, are waited after the completion of reacting, purifying can be obtained shown in formula (VI)
Trisaccharide.It can be used as lower step enzyme reaction receptor, and reaction equation is as shown in Figure 9.
In the specific embodiment of the application, the synthetic method of the tetrose shown in formula (VII) is：By formula (VI) institute
Trisaccharide, galactolipin (1.2~5.0 equivalent), adenosine triphyosphate (ATP) (1.2~5.0 equivalent), the uridine shown
Triphosphoric acid (UTP) (1.2~5.0 equivalent), MgCl2(5~100mmol), Tris-HCl buffer solutions (10~500mmol, pH 5.0
~10.0) then the pH value of reaction system is adjusted to 4.5~8.5, then adds galactokinase by obtained aqueous solution
(GalK), riboside generation enzyme (BLUSP) and β 1-4 galactosyl transferases (NmLgtB), reaction time are 3~72 hours, are treated
After the completion of reaction, purifying can obtain tetrose shown in formula (VII).It can be used as lower step enzyme reaction receptor, and reaction equation is as schemed
Shown in 10.
The another embodiment of the application provides a kind of synthetic method of core skeleton sugar chain 2,
(1) N-acetylglucosamine is coupled to β 1-3 glycosidic bonds using enzyme process modularization assembling 1 (as shown in Figure 4)
On tetrose shown in formula (VII), the pentasaccharides shown in formula (VIII) is synthesized；
(2) galactolipin is coupled to formula (VIII) institute with β 1-4 glycosidic bonds using enzyme process modularization assembling 2 (as shown in Figure 5)
On the pentasaccharides shown, six sugar shown in formula (IX) are synthesized；
Wherein, the R in formula (VII)~(IX)1Replace alkane for hydroxyl, azide substitution alkyl, alkynyl substituted alkyl, sulfydryl
Base, α-or beta configuration substitution alkyl, α-or beta configuration serine residue, α-or beta configuration threonine residues；
The enzyme that modified N-acetylglucosamine obtains intermediate 3 in the enzyme process modularization assembling 1 is N- acetylaminos
The enzyme of glucokinase and riboside generation enzyme, catalytic intermediary 3 and polysaccharide synthesis is shifted for β 1-3N- acetylglucosamines
Enzyme；
The enzyme that modified galactolipin obtains intermediate 4 in the enzyme process modularization assembling 1 is galactokinase and riboside life
Into enzyme, the enzyme of catalytic intermediary 4 and polysaccharide synthesis is β 1-4 galactosyl transferases.
Preferably, in the enzyme process modularization assembling 1, N-acetylglucosamine kinases is NahK, and riboside generates enzyme
For GlmU, β 1-3N- acetylglucosaminyl transferases are HpLgtA；
In the enzyme process modularization assembling 2, galactokinase GalK, riboside generates enzyme as BLUSP, β 1-4 galactolipins
Transferase is NmLgtB.
In the specific embodiment of the application, the synthetic method of the pentasaccharides shown in formula (VIII) is：By formula (VII)
(1.2~5.0 work as shown tetrose, N-acetylglucosamine (1.2~5.0 equivalent), adenosine triphyosphate (ATP)
Amount), uridine diphosphate guanosine triphosphate (UTP) (1.2~5.0 equivalent), MgCl2(5~100mmol), Tris-HCl buffer solutions (10~
500mmol, pH 5.0~10.0) obtained aqueous solution, then the pH value of reaction system is adjusted to 4.5~8.5, then adds N-
Acetylglucosamine kinases (NahK), UDP-N- acetylglucosamines generation enzyme (GlmU) and β 1-3N- acetylaminos Portugal
Grape sugar transferase (HpLgtA), reaction time are 3~72 hours, are waited after the completion of reacting, and purifying can be obtained shown in formula (VIII)
Pentasaccharides.It can be used as lower step enzyme reaction receptor, and reaction equation is as shown in figure 11.
In the specific embodiment of the application, the synthetic method of six sugar shown in formula (IX) is：By formula (VIII)
Shown pentasaccharides, galactolipin (1.2~5.0 equivalent), adenosine triphyosphate (ATP) (1.2~5.0 equivalent), uridine diphosphate
Guanosine triphosphate (UTP) (1.2~5.0 equivalent), MgCl2(5~100mmol), Tris-HCl buffer solutions (10~500mmol, pH
5.0~10.0) then the pH value of reaction system is adjusted to 4.5~8.5, then adds galactokinase by obtained aqueous solution
(GalK), riboside generation enzyme (BLUSP) and β 1-4 galactosyl transferases (NmLgtB), reaction time are 3~72 hours, are treated
After the completion of reaction, purifying can obtain six sugar shown in formula (IX).It can be used as lower step enzyme reaction receptor, reaction equation such as Figure 12
It is shown.
The application the third embodiment there is provided a kind of above method prepare the mono- aggressiveness of Lewis x, dimer or
Application in its sialylated derivative.
The 4th kind of the application is embodiment there is provided one kind containing LexThe sugar chain of single dimeric structure or dimeric structure
Fucose is coupled to α 1-3 glycosidic bonds shown in formula (VII) by synthetic method using enzyme process modularization assembling 3 (as shown in Figure 6)
Tetrose, the pentasaccharides shown in synthesis formula (I)；
Or, be coupled to fucose on six sugar shown in formula (IX) with α 1-3 glycosidic bonds using enzyme process modularization assembling 3, it closes
Into eight sugar shown in formula (II)；
Wherein, the R in formula (VII), formula (IX), formula (I)~(II)1For hydroxyl, azide substitution alkyl, alkynyl substituted alkane
Base, sulfydryl substitution alkyl, α-or beta configuration substitution alkyl, α-or beta configuration serine residue, α-or beta configuration threonine residues；
The enzyme that modified fucose obtains intermediate 5 in the enzyme process modularization assembling 3 is Fucokinase and riboside life
Into enzyme, the enzyme of catalytic intermediary 5 and polysaccharide synthesis is α 1-3 fucosyl transferases.
Preferably, in enzyme process modularization assembling 3, Fucokinase FKP, riboside generates enzyme as FKP, α 1-3 fucoses
Transferase is Hp α 1-3FucT.
In one embodiment of the invention, the synthetic method of the pentasaccharides shown in formula (I) is：By formula (VII) institute
Tetrose, fucose (1.2~5.0 equivalent), adenosine triphyosphate (ATP) (1.2~5.0 equivalent), the guanosine shown
Triphosphoric acid (GTP) (1.2~5.0 equivalent), MgCl2(5~100mmol), Tris-HCl buffer solutions (10~500mmol, pH 5.0
~10.0) then obtained aqueous solution is adjusted the pH value of reaction system to 4.5~8.5, then add Fucokinase (FKP),
Riboside generation enzyme (FKP) and α 1-3 fucosyl transferases (Hp α 1-3FucT), reaction time are 3~72 hours, wait to react
After the completion, purifying can obtain the pentasaccharides shown in formula (I).It can be used as lower step enzyme reaction receptor, reaction equation such as Figure 13 institutes
Show.
In one embodiment of the invention, the synthetic method of eight sugar shown in formula (II) is：By formula (IX) institute
Six sugar, fucose (1.2~5.0 equivalent), adenosine triphyosphate (ATP) (1.2~5.0 equivalent), the guanosine shown
Triphosphoric acid (GTP) (1.2~5.0 equivalent), MgCl2(5~100mmol), Tris-HCl buffer solutions (10~500mmol, pH 5.0
~10.0) then obtained aqueous solution is adjusted the pH value of reaction system to 4.5~8.5, then add Fucokinase (FKP),
Riboside generation enzyme (FKP) and α 1-3 fucosyl transferases (Hp α 1-3FucT), reaction time are 3~72 hours, wait to react
After the completion, purifying can obtain eight sugar shown in formula (II).It can be used as lower step enzyme reaction receptor, reaction equation such as Figure 14 institutes
Show
The 5th kind of the application is embodiment there is provided one kind containing sLexThe sugar chain of single dimeric structure or dimeric structure
Sialic acid is coupled to α 2-3 glycosidic bonds shown in formula (I) by synthetic method using enzyme process modularization assembling 4 (as shown in Figure 7)
Pentasaccharides, six sugar shown in parallel projects formula (III)；
Or, be coupled to sialic acid on eight sugar shown in formula (II) with α 2-3 glycosidic bonds using enzyme process modularization assembling 4, it closes
Into nine sugar shown in formula (IV)；
Wherein, the R in formula (I)~(IV)1For hydroxyl, azide substitution alkyl, alkynyl substituted alkyl, sulfydryl replace alkyl,
α-or beta configuration substitution alkyl, α-or beta configuration serine residue, α-or beta configuration threonine residues；
The enzyme that modified sialic acid obtains intermediate 6 in the enzyme process modularization assembling 4 is riboside generation enzyme, and catalysis is intermediate
Body 6 and the enzyme of polysaccharide synthesis are α 2-3 fucosyl transferases.
Preferably, in enzyme process modularization assembling 4, riboside generation enzyme is NmCSS, and α 2-3 sialyltransferases are
PmST1M144D。
In the specific embodiment of the application, the synthetic method of six sugar shown in formula (III) is：By formula (I) institute
Pentasaccharides, N-acetyl-neuraminate (Neu5Ac) (1.2~5.0 equivalent), the cytidine triphosphate (CTP) (1.2~5.0 shown
Equivalent), MgCl2(5~100mmol), Tris-HCl buffer solutions (10~500mmol, pH 5.0~10.0) obtained aqueous solution, so
The pH value of reaction system is adjusted to 4.5~8.5 afterwards, riboside generation enzyme (NmCSS) is then added and α 2-3 sialic acids turns
Enzyme (PmST1M144D) is moved, the reaction time is 0.5~36 hour, is waited after the completion of reacting, purifying can be obtained shown in formula (III)
Six sugar.Its reaction equation is as shown in figure 15.
In the specific embodiment of the application, the synthetic method of nine sugar shown in formula (IV) is：By formula (II) institute
Eight sugar, N-acetyl-neuraminate (Neu5Ac) (1.2~5.0 equivalent), the cytidine triphosphate (CTP) (1.2~5.0 shown
Equivalent), MgCl2(5~100mmol), Tris-HCl buffer solutions (10~500mmol, pH 5.0~10.0) obtained aqueous solution, so
The pH value of reaction system is adjusted to 4.5~8.5 afterwards, riboside generation enzyme (NmCSS) is then added and α 2-3 sialic acids turns
Enzyme (PmST1M144D) is moved, the reaction time is 0.5~36 hour, is waited after the completion of reacting, purifying can be obtained shown in formula (IV)
Nine sugar.Its reaction equation is as shown in figure 16.
In above-mentioned specific embodiment, react and use thin-layered chromatography (TLC) tracking reaction process after the completion, formula (I),
(III), (V)~(IX) solvents are EtOAc:MeOH:H2O:HOAc=4:2:1:0.2 and formula (II), (IV) synthesis expansion
Agent is nBuOH:MeOH:H2O:HOAc=2:2:1:1.
Used by the synthesis of above-mentioned polysaccharide in enzyme process modularization assembling strategy, reaction temperature is 0~37 DEG C, rotating speed for 0~
240r/min；The method of shutting down of the enzyme reaction is to add in the 4 DEG C absolute ethyl alcohols isometric with reaction solution simultaneously into reaction system
It is incubated 0~50 minute at 4 DEG C.
Chemical synthesis can be overcome the shortcomings of, but with regard to current enzyme with enzyme process modularization assembling strategy synthesis complex oligosaccharide
For the angle of source and practical application, the enzyme that synthesis of oligose chain is applied is broadly divided into two classes：One kind is glycosidase, the fermentoid
Abundance, performance are stable, reaction is simple, substrate is adaptable, have industrially been used at present, but the widow of this quasi-enzyme catalytic
Sugared synthesis yield is not good enough.Further, since the regioselectivity of the Transglycosylation of glucosides enzymatic is not high, and in synthetic reaction
Also there are the competitions of hydrolysis, cause product inhomogenous, it is difficult to isolate and purify, limit its extensive use.Second is sugar
Based transferase, the fermentoid have very high catalytic efficiency and stringent substrate selective.But the use of natural glycosyl transferase
There is also many limiting factors：The limited source of enzyme；Unstability, the complexity of enzyme cause to isolate and purify difficulty；Enzymatic
Glycosylation reaction expensive nucleosides activation sugar is needed to make glycosyl donor；Enzyme has stringent substrate specificity, to non-day
Right or abnormal substrate tolerance is poor.Disadvantage mentioned above significantly limits glycosidase and glycosyl transferase in enzyme process modularization
Synthesize the application in complex oligosaccharide.
Enzyme process module of the present invention generates three fermentoid of enzyme and sugared transferase containing sugared kinases, riboside, they
Efficient catalytic action, and mutually collaboration are played, forms an organic enzyme reaction system.To above-mentioned institute during experiment
Enzyme has carried out multiple optimization and screening, as a result, it has been found that：With NahK/GlmU, Bifidobacterium longum N-
Acetylhexosamine-1-kinase (NahK) and E.coli N-acetylglucosamine uridyltransferase
(GlmU), and 1-3-N-acetylglucosaminyltransferase (HpLgtA) of Helicobacter pylori β；
E.coli galactokinase(GalK),Bifidobacterium longum UDP-sugar pyrophosphorylase
(BLUSP) and 1-4-galactosyltransferase (NmLgtB) of Neisseria meningitides β；Neisseria
Meningitides CMP-sialic acid synthetase (NmCSS), and Pasteurella multocida
multifunctional 2–3-sialyltransferase 1M144D(PmST1M144D)；Bacteroides fragilis
Bifunctional L-fucokinase/GDP-fucose pyrophosphorylase (FKP) and Helicobacter
1-3-fucosyltransferase of pylori α (Hp α 1-3FucT) are used as enzyme of the present invention, and catalytic effect is best,
Combined coefficient is high, simple purification, and above-mentioned enzyme is bacterial origin, can in conventional escherichia expression system big scale
Up to purifying.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
Compound 1-9 mentioned in following embodiments corresponds respectively to R1General formula V-IX, I- for azide substitution alkyl
The compound of IV.
Embodiment 1：LexAnd sLexSingle aggressiveness, dimer oligosaccharides synthesis
Step is as follows：
(1) (the Gal β 1,4Glc β ProN of lactoside 1 of chemical method synthesis beta configuration3)
Lactose 10 (10g, 29.23mmol), aceticanhydride (55mL) and sodium acetate (9.6g) are added in into 500mL round-bottomed flasks,
It is refluxed under 160 DEG C of reaction conditions 6 hours.Thin-layer chromatography (PE:EA=1:1) detection is after the reaction was complete, concentrated by rotary evaporation.Institute
It obtaining solid to redissolve in 250mL dichloromethane, be extracted twice with semi-saturation saline solution, saturated sodium bicarbonate solution extracts three times,
Double steaming solution extracts three times, detaches organic phase later, then dry organic phase with anhydrous sodium sulfate, concentrated by rotary evaporation obtains yellowish
Color solid chemical compound 11 (18.70g, 94%).
Compound 11 (1.0g, 1.47mmol), dichloromethane are added in into the special reaction tube of microwave that volume is 10mL
(5.0mL), boron trifluoride ether (0.36mL) and trimethylene chlorohydrin (0.25mL), magnetic agitation, pre- stirring 2 minutes, reaction temperature
It is 50 DEG C to spend, and the reaction time is 15 minutes.It is parallel to open 5 groups according to identical reaction condition.Reaction solution is collected, is quenched with triethylamine
It goes out after reaction, concentrated by rotary evaporation, Flash silica column separating purification obtains faint yellow solid compound 12 (2.75g, 53%).
Into 250mL round-bottomed flasks add in compound 12 (2.40g, 3.37mmol), N,N-dimethylformamide (40mL),
Sodium azide (1.2g) and tetrabutylammonium iodide (0.12g), 110 DEG C are stirred at reflux 12 hours.Thin-layer chromatography (PE:EA=1:1) it examines
It surveys after the reaction was complete, is filtered using diatomite, then concentrated by rotary evaporation, Flash silica column separating purification, obtain faint yellow solid chemical combination
Object 13 (1.93g, 90%).
Compound 13 (1.50g, 2.36mmol), methanol (10mL), sodium methoxide are added in into 50mL round-bottomed flasks, until molten
Liquid system pH value arrives 9-10 or so, is stirred at room temperature 4 hours, thin-layer chromatography detection (EtOAc:MeOH:H2O:HOAc=4:2:1:
0.2) after the reaction was complete, the pH value of reaction solution is adjusted to neutrality, concentrated by rotary evaporation, Flash silica column with acid-exchange resin
It isolates and purifies, obtains compound as white solid 1 (0.90g, 90%).
The synthetic route of compound 1 is as shown in Figure 8.
(2) enzyme process module assembled 1 synthesizes three sugar compounds, 2 [GlcNAc β (l-3) Gal β (l-4) Glc β ProN3]
[GlcNAc β (l-3) Gal β (l-4) the Glc β ProN of the compound of the present invention 23] synthetic method it is as follows：
By lactose acceptor compound 1 (80mg), N-acetylglucosamine (54mg), ATP (124mg), UTP (118mg),
Tris-HCl buffer solutions (100mmol, pH 8.0) and MgCl2(20mmol) (Tris and MgCl2Dosage by end reaction liquid
Volume is determining to calculate) it is dissolved in 50mL centrifuge tubes, NahK/GlmU (1.0-3.0mg) and HpLgtA (0.5-2.0mg) is added in,
After adding distilled water to total volume 10mL, reaction system is placed in shaking table, 37 DEG C, 140r/min be incubated 16 hours.Thin-layer chromatography
(EtOAc:MeOH:H2O:EtOH=4:2:1:0.2) after the completion of detection reaction, the anhydrous second isometric with reaction system is added in
Alcohol stands 30 minutes to terminate reaction in 4 DEG C of refrigerators.Then 4 DEG C of reaction system, 12000r/min are centrifuged 20 minutes, collected
Supernatant, concentrated by rotary evaporation by Flash silica post separation, obtain white compound 2 (105mg, 89%).Parameter is as follows：1H
NMR(600MHz,D2O) δ 4.70 (d, J=8.4Hz, 1H), 4.50 (d, J=7.8Hz, 1H), 4.45 (d, J=7.8Hz, 1H),
4.16 (d, J=3.0Hz, 1H), 4.03-3.46 (m, 20H), 3.32 (t, J=8.4Hz, 1H), 2.05 (s, 3H), 1.93 (p, J
=6.6Hz, 2H)；13C NMR(151MHz,D2O)δ174.95,102.92,102.84,102.10,81.96,78.34,75.64,
74.88,74.76,74.35,73.55,72.79,70.00,69.68,68.33,67.36,60.96,60.47,60.06,
55.65,47.87,28.23,22.19。
The synthetic route of compound 2 is as shown in Figure 9.
(3) enzyme process module assembled 2 synthesizes four sugar compounds, 3 [Gal β (l-4) GlcNAc β (l-3) Gal β (l-4) Glc β
ProN3]
[Gal β (l-4) GlcNAc β (l-3) Gal β (l-4) the Glc β ProN of the compound of the present invention 33] synthetic method such as
Under：
By three sugar compounds 2 (80mg), galactolipin (30mg), ATP (84mg), UTP (80mg), Tris-HCl buffer solutions
(100mmol, pH 7.5) and MgCl2(20mmol) (Tris and MgCl2Dosage calculated by the volume of end reaction liquid it is determining)
It is dissolved in 50mL centrifuge tubes, adds in GalK (2.0-4.0mg), BLUSP (2.0-5.0mg) and NmLgtB (1.0-3.0mg), add double
Steam water to total volume 10mL after, reaction system is placed in shaking table, 37 DEG C, 140r/min be incubated 12 hours.Thin-layer chromatography
(EtOAc:MeOH:H2O:EtOH=4:2:1:0.2) after the completion of detection reaction, the anhydrous second isometric with reaction system is added in
Alcohol makes enzyme denaturation in 30 minutes to terminate reaction in 4 DEG C of refrigerators standings.Then 4 DEG C of reaction system, 12000r/min are centrifuged 20 points
Clock, collects supernatant, and concentrated by rotary evaporation by the quick post separation of silicagel column, obtains white compound 3 (94mg, 93%).Parameter is such as
Under：1H NMR(600MHz,D2O) δ 4.73 (d, J=8.4Hz, 1H), 4.50 (d, J=7.8Hz, 1H), 4.49 (d, J=7.8Hz,
1H), 4.46 (d, J=8.4Hz, 1H), 4.17 (d, J=3.6Hz, 1H), 4.03-3.96 (m, 3H), 3.94 (d, J=3.0Hz,
1H), 3.87 (dd, J=4.8,12.6Hz, 1H), 3.84-3.54 (m, 19H), 3.48 (t, J=6.6Hz, 2H), 3.33 (t, J=
8.4Hz, 1H), 2.05 (s, 3H), 1.93 (p, J=6.6Hz, 2H)；13C NMR(151MHz,D2O)δ174.89,102.92,
102.84,102.73,102.09,82.05,78.35,78.14,75.33,74.87,74.75,74.53,74.34,72.79,
72.49,72.16,70.96,69.95,68.55,68.31,67.36,61.04,60.96,60.06,59.85,55.18,
47.87,28.24,22.22。
The synthetic route of compound 3 is as shown in Figure 10.
(4) enzyme process modularization 1 synthesizes five sugar compounds, 4 [GlcNAc β (l-3) Gal β (l-4) GlcNAc β (l-3) Gal β
(l-4)GlcβProN3]
[GlcNAc β (l-3) Gal β (l-4) GlcNAc β (l-3) Gal β (l-4) the Glc β ProN of the compound of the present invention 43]
Synthetic method it is as follows：
By four sugar compounds 3 (150mg), N-acetylglucosamine (54.6mg), ATP (135.9mg), UTP
(119.4mg), Tris-HCl buffer solutions (100mmol, pH 8.0) and MgCl2(20mmol) (Tris and MgCl2Dosage by most
The volume of end reaction liquid is determining to calculate) it is dissolved in 50mL centrifuge tubes, add in NahK/GlmU (1.0-3.0mg) and HpLgtA
(0.5-2.0mg) after adding distilled water to total volume 10mL, reaction system is placed in shaking table, 37 DEG C, 140r/min be incubated it is 16 small
When.Thin-layer chromatography (EtOAc:MeOH:H2O:EtOH=4:2:1:0.2) after the completion of detection reaction, the bodies such as addition and reaction system
Long-pending absolute ethyl alcohol makes enzyme denaturation in 30 minutes to terminate reaction in 4 DEG C of refrigerators standings.Then by 4 DEG C of reaction system, 12000r/
Min is centrifuged 20 minutes, collects supernatant, and concentrated by rotary evaporation by the quick post separation of silicagel column, obtains white compound 4
(167.8mg, 89%).Parameter is as follows：1H NMR(600MHz,D2O) δ 4.67 (dd, J=11.6,8.4Hz, 2H), 4.46 (dd, J
=12.0,7.9Hz, 2H), 4.42 (d, J=7.9Hz, 1H), 4.13 (d, J=3.0Hz, 2H), 4.00-3.87 (m, 4H), 3.82
(dd, J=4.2,12Hz, 1H), 3.80-3.40 (m, 26H), 3.29 (m, J=8.4Hz, 1H), 2.02 (d, J=3.0Hz, 6H),
1.90 (p, J=6.6Hz, 2H)；13C NMR(151MHz,D2O)δ174.87,174.82,102.89,102.84,102.81,
102.69,102.07,81.98,81.94,78.31,78.13,75.61,74.84,74.82,74.72,74.50,74.31,
73.51,72.75,72.12,69.95,69.91,69.64,68.28,67.31,60.92,60.45,60.02,59.82,
55.62,55.10,47.84,28.21,22.17。
The synthetic route of compound 4 is as shown in figure 11.
(5) enzyme process module assembled 2 synthesizes six sugar compounds 5
[Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcNAcβ(l-3)Galβ(l-4)GlcβProN3]
[Gal β (l-4) GlcNAc β (l-3) Gal β (l-4) GlcNAc β (l-3) the Gal β (l-4) of the compound of the present invention 5
GlcβProN3] synthetic method it is as follows：
By five sugar compounds 4 (150mg), galactolipin (32.6mg), ATP (99.8mg), UTP (87.7mg), Tris-HCl
Buffer solution (100mmol, pH 7.5) and MgCl2(20mmol) (Tris and MgCl2Dosage counted by the volume of end reaction liquid
Calculate and determine) it is dissolved in 50mL centrifuge tubes, add in GalK (2.0-4.0mg), BLUSP (2.0-5.0mg) and NmLgtB (1.0-
3.0mg), after adding distilled water to total volume 10mL, reaction system is placed in shaking table, 37 DEG C, 140r/min be incubated 12 hours.It is thin
Layer chromatography (EtOAc:MeOH:H2O:EtOH=4:2:1:0.2) it after the completion of detection reaction, adds in and the isometric nothing of reaction system
Water-ethanol makes enzyme denaturation in 30 minutes to terminate reaction in 4 DEG C of refrigerators standings.Then 4 DEG C of reaction system, 12000r/min are centrifuged
20 minutes, collect supernatant, concentrated by rotary evaporation, by the quick post separation of silicagel column, obtain white compound 5 (160.5mg,
92%).Parameter is as follows：1H NMR(600MHz,D2O) δ 4.65 (d, J=7.8Hz, 2H), 4.45 (d, J=7.2Hz, 1H), 4.43
(d, J=6.6Hz, 1H), 4.42 (d, J=7.8Hz, 1H), 4.39 (d, J=7.8Hz, 1H), 4.11 (d, J=3.0Hz, 2H),
3.98-3.48 (m, 35H), 3.42 (t, J=6.6Hz, 2H), 3.27 (t, J=8.4Hz, 1H), 1.99 (s, 6H), 1.87 (p, J
=6.6Hz, 2H)；13C NMR(151MHz,D2O)δ174.77,102.81,102.75,102.72,102.65,102.63,
101.98,81.94,81.90,78.17,77.97,75.22,74.74,74.64,74.41,74.22,72.65,72.36,
72.04,70.83,69.82,68.41,68.20,67.23,60.91,60.83,59.90,59.70,55.05,55.01,
47.73,28.10,22.04。
The synthetic route of compound 5 is as shown in figure 12.
(6) enzyme process module assembled 3 synthesizes five sugar compounds, 6 [Gal β (l-4) (Fuc α 1-3) GlcNAc β (l-3) Gal β (l-
4)GlcβProN3]
[Gal β (l-4) (Fuc α 1-3) GlcNAc β (l-3) Gal β (l-4) the Glc β ProN of the compound of the present invention 63] conjunction
It is as follows into method：
By four sugar compounds 3 (160mg), fucose (49mg), ATP (152mg), GTP (180mg), Tris-HCl bufferings
Liquid (100mmol, pH 7.5) and MgCl2(20mmol) (Tris and MgCl2Dosage by the volume of end reaction liquid come calculate really
It is fixed) it is dissolved in 50mL centrifuge tubes, FKP (2.0-4.0mg), Hp α 1-3FucT (2.0-5.0mg) are added in, adds distilled water to total volume
After 10mL, reaction system is placed in shaking table, 37 DEG C, 140r/min be incubated 15 hours.Thin-layer chromatography (EtOAc:MeOH:H2O:
EtOH=4:2:1:0.2) after the completion of detection reaction, addition and the isometric absolute ethyl alcohol of reaction system stand 30 in 4 DEG C of refrigerators
Minute makes enzyme denaturation to terminate reaction.Then 4 DEG C of reaction system, 12000r/min are centrifuged 20 minutes, collects supernatant, revolving
Concentration by the quick post separation of silicagel column, obtains white compound 6 (174mg, 92%).Parameter is as follows：1H NMR(600MHz,
D2O) δ 5.12 (d, J=3.6Hz, 1H), 4.84 (m, 1H), 4.70 (d, J=8.4Hz, 1H), 4.48 (d, J=7.8Hz, 1H),
4.45 (d, J=7.8Hz, 1H), 4.42 (d, J=8.4Hz, 1H), 4.15 (d, J=3.0Hz, 1H), 4.05-3.56 (m, 26H),
3.48 (dd, J=7.9,9.6Hz, 1H), 3.46 (t, J=6.6Hz, 2H), 3.30 (t, J=8.5Hz, 1H), 2.02 (s, 3H),
1.91 (p, J=6.5Hz, 2H), 1.17 (d, J=6.6Hz, 3H)；13C NMR(151MHz,D2O)δ174.58,102.84,
102.43,102.01,101.67,98.51,81.97,78.24,75.01,74.80,74.76,74.66,74.26,72.96,
72.69,72.38,71.81,70.95,69.85,69.10,68.25,68.20,67.61,67.26,66.59,61.40,
60.87,59.97,59.54,55.86,47.78,28.15,22.18,15.25。
The synthetic route of compound 6 is as shown in figure 13.
(7) enzyme process module assembled 3 synthesizes eight sugar compounds, 7 [Gal β (l-4) (Fuc α 1-3) GlcNAc β (l-3) Gal β (l-
4)(Fucα1-3)GlcNAcβ(l-3)Galβ(l-4)GlcβProN3]
[Gal β (l-4) (Fuc α 1-3) GlcNAc β (l-3) the Gal β (l-4) (Fuc α 1-3) of the compound of the present invention 7
GlcNAcβ(l-3)Galβ(l-4)GlcβProN3] synthetic method it is as follows：
By six sugar compounds 5 (80mg), fucose (17mg), ATP (52.7mg), GTP (61.8mg), Tris-HCl bufferings
Liquid (100mmol, pH 7.5) and MgCl2(20mmol) (Tris and MgCl2Dosage by the volume of end reaction liquid come calculate really
It is fixed) it is dissolved in 50mL centrifuge tubes, FKP (2.0-4.0mg), Hp α 1-3FucT (2.0-5.0mg) are added in, adds distilled water to total volume
After 10mL, reaction system is placed in shaking table, 37 DEG C, 140r/min be incubated 15 hours.Thin-layer chromatography (nBuOH:MeOH:H2O:
EtOH=2:2:1:1) after the completion of detection reaction, addition and the isometric absolute ethyl alcohol of reaction system stand 30 points in 4 DEG C of refrigerators
Clock makes enzyme denaturation to terminate reaction.Then 4 DEG C of reaction system, 12000r/min are centrifuged 20 minutes, collects supernatant, revolving is dense
Contracting by the quick post separation of silicagel column, obtains white compound 7 (88.2mg, 88%).Parameter is as follows：1H NMR(600MHz,
D2O) δ 5.13 (d, J=3.6Hz, 1H), 5.11 (d, J=4.2Hz, 1H), 4.72 (d, J=5.4Hz, 1H), 4.70 (d, J=
8.4Hz, 1H), 4.49 (d, J=7.8Hz, 1H), 4.47 (d, J=7.8Hz, 1H), 4.44 (t, J=7.2Hz, 2H), 4.15 (d,
J=3.0Hz, 1H), 4.10 (d, J=3.0Hz, 1H), 4.02-3.48 (m, 43H), 3.46 (t, J=6.6Hz, 2H), 3.31 (t,
J=8.4Hz, 1H), 2.02 (s, 3H), 2.02 (s, 3H), 1.91 (p, J=6.6Hz, 2H), 1.18 (d, J=6.6Hz, 3H),
1.15 (d, J=6.6Hz, 3H)；13C NMR(151MHz,D2O)δ174.69,174.63,102.92,102.50,102.47,
102.09,101.72,101.71,98.68,98.57,82.04,81.59,78.31,75.07,75.04,74.89,74.84,
74.74,74.42,74.33,73.00,72.77,72.74,72.45,71.89,71.83,71.03,70.50,69.93,
69.17,69.15,68.33,68.28,68.21,67.68,67.62,67.35,66.66,61.49,61.44,60.93,
60.04,59.61,59.42,55.93,47.85,28.21,22.23,15.30,15.26。
The synthetic route of compound 7 is as shown in figure 14.
(8) enzyme process module assembled 4 synthesizes six sugar compounds 8
[Neu5Acα(2-3)Galβ(l-4)(Fucα1-3)GlcNAcβ(l-3)Galβ(l-4)GlcβProN3]
[Neu5Ac α (2-3) Gal β (l-4) (Fuc α 1-3) GlcNAc β (l-3) the Gal β (l-4) of the compound of the present invention 8
GlcβProN3]
Synthetic method is as follows：
By five sugar compounds 6 (80mg), Neu5Ac (22mg), CTP (37.6mg), Tris-HCl buffer solutions (100mmol,
PH 8.0) and MgCl2(20mmol) (Tris and MgCl2Dosage calculated by the volume of end reaction liquid it is determining) be dissolved in 50mL
In centrifuge tube, NmCSS (2.0-4.0mg), PmST1M144D (2.0-5.0mg) are added in, it, will after adding distilled water to total volume 10mL
Reaction system is placed in shaking table, 37 DEG C, 140r/min be incubated 3 hours.Thin-layer chromatography (EtOAc:MeOH:H2O:EtOH=4:2:
1:0.2) after the completion of detection reaction, addition and the isometric absolute ethyl alcohol of reaction system, standing 30 minutes in 4 DEG C of refrigerators becomes enzyme
Property with terminate reaction.Then 4 DEG C of reaction system, 12000r/min are centrifuged 20 minutes, collects supernatant, concentrated by rotary evaporation passes through
The quick post separation of silicagel column obtains white compound 8 (62.5mg, 62%).Parameter is as follows：1H NMR(600MHz,D2O)δ5.08
(d, J=4.2Hz, 1H), 4.67 (d, J=7.8Hz, 1H), 4.49 (d, J=7.8Hz, 1H), 4.45 (d, J=8.4Hz, 1H),
4.40 (d, J=7.8Hz, 1H), 4.13 (d, J=3.0Hz, 1H), 4.05 (dd, J=2.6,9.8Hz, 1H), 4.04 (d, J=
2.4Hz, 1H), 4.00-3.52 (m, 33H), 3.49 (t, J=8.8Hz, 1H), 3.43 (t, J=6.6Hz, 2H), 3.30 (t, J=
8.4Hz, 1H), 2.73 (dd, J=4.2,12.6Hz, 1H), 2.00 (s, 3H), 1.99 (s, 3H), 1.88 (p, J=6.6Hz,
2H), 1.76 (t, J=12.0Hz, 1H), 1.13 (d, J=6.6Hz, 3H)；13CNMR(151MHz,D2O)δ174.88,174.55,
173.74,102.81,102.47,101.97,101.42,99.51,98.46,81.97,78.19,75.52,74.89,74.79,
74.76,74.64,74.51,74.23,72.88,72.78,72.65,71.77,71.73,69.82,69.13,69.04,
68.17,67.96,67.57,67.23,67.18,66.53,62.46,61.37,60.85,59.92,59.37,59.18,
55.83,51.56,47.73,39.65,28.11,22.12,21.90,15.16。
The synthetic route of compound 8 is as shown in figure 15.
(9) enzyme process module assembled 4 synthesizes nine sugar compounds 9
[Neu5Acα(2-3)Galβ(l-4)(Fucα1-3)GlcNAcβ(l-3)Galβ(l-4)(Fucα1-3)GlcNAcβ
(l-3)Galβ(l-4)GlcβProN3]
[Neu5Ac α (2-3) Gal β (l-4) (Fuc α 1-3) GlcNAc β (l-3) the Gal β (l-4) of the compound of the present invention 9
(Fucα1-3)Glc-NAcβ(l-3)Galβ(l-4)GlcβProN3] synthetic method it is as follows：
By eight sugar compounds 7 (50mg), Neu5Ac (16mg), CTP (27.3mg), Tris-HCl buffer solutions (100mmol,
PH 8.0) and MgCl2(20mmol) (Tris and MgCl2Dosage calculated by the volume of end reaction liquid it is determining) be dissolved in 50mL
In centrifuge tube, NmCSS (2.0-4.0mg), PmST1M144D (2.0-5.0mg) are added in, it, will after adding distilled water to total volume 10mL
Reaction system is placed in shaking table, 37 DEG C, 140r/min be incubated 3 hours.Thin-layer chromatography (nBuOH:MeOH:H2O:EtOH=2:2:
1:1) after the completion of detection reaction, addition and the isometric absolute ethyl alcohol of reaction system, standing 30 minutes in 4 DEG C of refrigerators makes enzyme denaturation
To terminate reaction.Then 4 DEG C of reaction system, 12000r/min are centrifuged 20 minutes, collects supernatant, concentrated by rotary evaporation passes through silicon
The quick post separation of rubber column gel column obtains white compound 9 (32.8mg, 50%).Parameter is as follows：1H NMR(600MHz,D2O)δ5.07
(dd, J=7.7,3.9Hz, 2H), 4.65 (t, J=8.6Hz, 2H), 4.48 (d, J=7.8Hz, 1H), 4.44 (d, J=8.0Hz,
1H), 4.39 (t, J=7.4Hz, 2H), 4.11 (d, J=3.2Hz, 1H), 4.06-4.02 (m, 2H), 3.98-3.38 (m, 51H),
3.26 (dd, J=10.6,6.2Hz, 1H), 2.71 (dd, J=12.7,4.7Hz, 1H), 2.00 (s, 9H) 1.86 (p, J=
6.6Hz, 2H), 1.75 (t, J=12.1Hz, 1H), 1.11 (dd, J=12.4,6.5Hz, 6H)；13C NMR(151MHz,D2O)δ
174.65,174.57,102.86,102.48,102.04,101.65,101.45,99.57,98.65,98.50,82.00,
81.56,78.21,75.58,75.02,74.90,74.85,74.80,74.70,74.57,74.39,74.27,72.92,
72.83,72.71,72.64,71.84,71.79,71.68,70.45,69.89,69.20,69.09,68.24,68.17,
68.00,67.63,67.55,67.29,66.62,66.59,62.51,61.44,60.89,59.96,59.54,59.42,
55.88,51.77,51.61,47.78,46.56,39.70,28.16,22.18,21.97,15.35,15.22。
The synthetic route of compound 9 is as shown in figure 16.
The foregoing is merely the preferred embodiments of the application, are not limited to the application, for the skill of this field
For art personnel, the application can have various modifications and variations.It is all within spirit herein and principle, made any repair
Change, equivalent replacement, improvement etc., should be included within the protection domain of the application.
Claims (10)
1. a kind of synthetic method of core skeleton sugar chain 1, it is characterized in that,
1) N-acetylglucosamine β 1-3 glycosidic bonds are coupled on the disaccharides shown in formula (V) using enzyme process modularization assembling 1,
Trisaccharide shown in synthesis formula (VI)；
2) galactolipin is coupled to β 1-4 glycosidic bonds on the trisaccharide shown in formula (VI) using enzyme process modularization assembling 2, synthesizes formula
(VII) tetrose shown in；
Wherein, the R in formula (V)~(VII)1For hydroxyl, azide substitution alkyl, alkynyl substituted alkyl, sulfydryl replace alkyl, α-or
Beta configuration substitution alkyl, α-or beta configuration serine residue, α-or beta configuration threonine residues；
The enzyme that modified N- acetylamino c glucose obtains intermediate 1 in the enzyme process modularization assembling 1 is N- acetamido glucoses
The enzyme of sugared kinases and riboside generation enzyme, catalytic intermediary 1 and polysaccharide synthesis is β 1-3N- acetylglucosaminyl transferases；
The enzyme that modified galactolipin obtains intermediate 2 in the enzyme process modularization assembling 1 is that galactokinase and riboside generate enzyme,
Catalytic intermediary 2 and the enzyme of polysaccharide synthesis are β 1-4 galactosyl transferases.
2. synthetic method as described in claim 1, it is characterized in that, in the enzyme process modularization assembling 1, N- acetamido glucoses
Sugared kinases is NahK, and riboside generation enzyme is GlmU, and β 1-3N- acetylglucosaminyl transferases are HpLgtA；
In the enzyme process modularization assembling 2, galactokinase GalK, riboside generation enzyme is BLUSP, and β 1-4 galactolipins shift
Enzyme is NmLgtB；
Preferably, the synthesis of the disaccharides shown in formula (V) uses following methods：After lactose is reacted with aceticanhydride, by its whole
Exposed hydroxyl is protected with acetyl group；Then full acetylated lactose is subjected to beta configuration glycosylation reaction using microwave method, finally
Azide and deprotection successively, you can obtain；
Preferably, the synthetic method of the trisaccharide shown in formula (VI) is：By shown in formula (V) lactoside, N-acetylglucosamine,
Adenosine triphyosphate uridine diphosphate guanosine triphosphate, MgCl2, Tris-HCl buffer aqueous solutions, then by reactant
The pH value of system is adjusted to 4.5~8.5, then adds NahK, GlmU and HpLgtA, the reaction time is 3~72 hours, waits to react
After the completion, purifying can obtain the trisaccharide shown in formula (VI)；
Preferably, the synthetic method of the tetrose shown in formula (VII) is：By trisaccharide, galactolipin, the adenosine shown in formula (VI)
Triphosphoric acid, uridine diphosphate guanosine triphosphate, MgCl2, Tris-HCl buffer aqueous solutions, then by the pH value tune of reaction system
Then section adds GalK, BLUSP and NmLgtB, the reaction time is 3~72 hours, is waited after the completion of reacting, pure to 4.5~8.5
Change can obtain tetrose shown in formula (VII).
3. a kind of synthetic method of core skeleton sugar chain 2, it is characterized in that,
(1) N-acetylglucosamine is coupled to four shown in formula (VII) using enzyme process modularization assembling 1 with β 1-3 glycosidic bonds
On sugar, the pentasaccharides shown in formula (VIII) is synthesized；
(2) galactolipin is coupled to β 1-4 glycosidic bonds on the pentasaccharides shown in formula (VIII) using enzyme process modularization assembling 2, synthesized
Six sugar shown in formula (IX)；
Wherein, the R in formula (VII)~(IX)1For hydroxyl, azide substitution alkyl, alkynyl substituted alkyl, sulfydryl replace alkyl, α-or
Beta configuration substitution alkyl, α-or beta configuration serine residue, α-or beta configuration threonine residues；
The enzyme that modified N-acetylglucosamine obtains intermediate 3 in the enzyme process modularization assembling 1 is N- acetamido glucoses
The enzyme of sugared kinases and riboside generation enzyme, catalytic intermediary 3 and polysaccharide synthesis is β 1-3N- acetylglucosaminyl transferases；
The enzyme that modified galactolipin obtains intermediate 4 in the enzyme process modularization assembling 1 is that galactokinase and riboside generate enzyme,
Catalytic intermediary 4 and the enzyme of polysaccharide synthesis are β 1-4 galactosyl transferases.
4. synthetic method as claimed in claim 3, it is characterized in that, in the enzyme process modularization assembling 1, N- acetamido glucoses
Sugared kinases is NahK, and riboside generation enzyme is GlmU, and β 1-3N- acetylglucosaminyl transferases are HpLgtA；
In the enzyme process modularization assembling 2, galactokinase GalK, riboside generation enzyme is BLUSP, and β 1-4 galactolipins shift
Enzyme is NmLgtB；
Preferably, the synthetic method of the pentasaccharides shown in formula (VIII) is：By tetrose, the N- acetamido glucoses shown in formula (VII)
Sugar, adenosine triphyosphate, uridine diphosphate guanosine triphosphate, MgCl2, Tris-HCl buffer aqueous solutions, then will be anti-
The pH value of system is answered to adjust to 4.5~8.5, then adds NahK, GlmU and HpLgtA, the reaction time is 3~72 hours, is treated
After the completion of reaction, purifying can obtain the pentasaccharides shown in formula (VIII)；
Preferably, the synthetic method of six sugar shown in formula (IX) is：By pentasaccharides, galactolipin, the adenine core shown in formula (VIII)
Guanosine triphosphate, uridine diphosphate guanosine triphosphate, MgCl2, Tris-HCl buffer aqueous solutions, then by the pH value of reaction system
It adjusting to 4.5~8.5, then adds GalK, BLUSP and NmLgtB, the reaction time is 3~72 hours, is waited after the completion of reacting,
Purifying can obtain six sugar shown in formula (IX).
5. a kind of any synthetic method of Claims 1 to 4 is preparing the mono- aggressiveness of Lewis x, dimer or its sialic acid
Change the application in derivative.
6. one kind contains LexThe synthetic method of the sugar chain of single dimeric structure or dimeric structure, it is characterized in that, utilize enzyme process modularization
Fucose is coupled to tetrose shown in formula (VII), the pentasaccharides shown in synthesis formula (I) by assembling 3 with α 1-3 glycosidic bonds；
Or, be coupled to fucose on six sugar shown in formula (IX) with α 1-3 glycosidic bonds using enzyme process modularization assembling 3, synthesize formula
(II) eight sugar shown in；
Wherein, the R in formula (VII), formula (IX), formula (I)~(II)1For hydroxyl, azide substitution alkyl, alkynyl substituted alkyl, sulfydryl
Replace alkyl, α-or beta configuration substitution alkyl, α-or beta configuration serine residue, α-or beta configuration threonine residues；
The enzyme that modified fucose obtains intermediate 5 in the enzyme process modularization assembling 3 is that Fucokinase and riboside generate enzyme,
Catalytic intermediary 5 and the enzyme of polysaccharide synthesis are α 1-3 fucosyl transferases.
7. synthetic method as claimed in claim 6, it is characterized in that, in enzyme process modularization assembling 3, Fucokinase FKP, sugar
Nucleosides generation enzyme is FKP, and α 1-3 fucosyl transferases are Hp α 1-3FucT；
Preferably, the synthetic method of the pentasaccharides shown in formula (I) is：By tetrose, fucose, the adenosine shown in formula (VII)
Triphosphoric acid, guanopterin nucleoside triphosphate, MgCl2, Tris-HCl buffer aqueous solutions, then by the pH value tune of reaction system
Then section adds FKP and Hp α 1-3FucT, the reaction time is 3~72 hours, is waited after the completion of reacting, and is purified to 4.5~8.5
The pentasaccharides shown in formula (I) can be obtained；
Preferably, the synthetic method of eight sugar shown in formula (II) is：By six sugar, fucose, the adenosine shown in formula (IX)
Triphosphoric acid, guanopterin nucleoside triphosphate, MgCl2(5~100mmol), Tris-HCl buffer aqueous solutions, then will reaction
The pH value of system is adjusted to 4.5~8.5, then adds FKP and Hp α 1-3FucT, the reaction time is 3~72 hours, waits to react
After the completion, purifying can obtain eight sugar shown in formula (II).
8. one kind contains sLexThe synthetic method of the sugar chain of single dimeric structure or dimeric structure, it is characterized in that, utilize enzyme process modularization
Sialic acid is coupled to pentasaccharides shown in formula (I) by assembling 4 with α 2-3 glycosidic bonds, six sugar shown in parallel projects formula (III)；
Or, be coupled to sialic acid on eight sugar shown in formula (II) with α 2-3 glycosidic bonds using enzyme process modularization assembling 4, synthesize formula
(IV) nine sugar shown in；
Wherein, the R in formula (I)~(IV)1For hydroxyl, azide substitution alkyl, alkynyl substituted alkyl, sulfydryl replace alkyl, α-or β-
Configuration substitution alkyl, α-or beta configuration serine residue, α-or beta configuration threonine residues；
The enzyme that modified sialic acid obtains intermediate 6 in the enzyme process modularization assembling 4 is riboside generation enzyme, catalytic intermediary 6
Enzyme with polysaccharide synthesis is α 2-3 fucosyl transferases.
9. synthetic method as claimed in claim 8, it is characterized in that, in enzyme process modularization assembling 4, riboside generation enzyme is
NmCSS, α 2-3 sialyltransferases are PmST1M144D；
Preferably, the synthetic method of six sugar shown in formula (III) is：By shown in formula (I) pentasaccharides, N-acetyl-neuraminate, born of the same parents
Pyrimidine nucleoside triphosphoric acid, MgCl2, Tris-HCl buffer aqueous solutions, then the pH value of reaction system is adjusted to 4.5~
8.5, NmCSS and PmST1M144D is then added, the reaction time is 0.5~36 hour, is waited after the completion of reacting, purifying can obtain
Obtain six sugar shown in formula (III)；
Preferably, the synthetic method of nine sugar shown in formula (IV) is：By shown in formula (II) eight sugar, N-acetyl-neuraminate, born of the same parents
Pyrimidine nucleoside triphosphoric acid, MgCl2, Tris-HCl buffer aqueous solutions, then the pH value of reaction system is adjusted to 4.5~
8.5, NmCSS and PmST1M144D is then added, the reaction time is 0.5~36 hour, is waited after the completion of reacting, purifying can obtain
Obtain nine sugar shown in formula (IV).
10. the synthetic method as described in claim 1~9 is any, it is characterized in that, the condition of the enzyme process modularization assembling of use
For reaction temperature is 0~37 DEG C, and rotating speed is 0~240r/min；The method of shutting down of the enzyme reaction is added in into reaction system
It 4 DEG C of isometric absolute ethyl alcohols and is incubated 0~50 minute at 4 DEG C with reaction solution.
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